Dendritic spines are small (0.1 - 0.01 femtolitter) excitatory postsynaptic compartments emanating from the dendritic surface. Ca2+ influx into spines activates signaling networks consisting of hundreds of species of proteins that induce diverse forms of synaptic plasticity. Rho GTPase proteins, particularly Rac1, RhoA and Cdc42, are critical components of these signaling networks, and their activation plays an important role in regulating the morphology and function of dendritic spines. Consistent with the important role of Rho signaling in spine morphology and function, mutations in Rho signaling pathways are associated with many forms of mental retardation and autism. In this study, we will develop a technique to measure Rho signaling in single dendritic spines while they undergo morphological and functional plasticity in brain slices. To do so, we will combine 2-photon fluorescence lifetime imaging microscopy (2pFLIM) with fluorescent resonance energy transfer-based Rho activity sensors extensively optimized for 2pFLIM. Our preliminary data demonstrates that the activity of Cdc42 is restricted to spines undergoing synaptic plasticity, while Rac1 and RhoA activation spreads along dendrites over ~10 5m and invades neighboring spines. These results suggest that each Rho signaling pathway functions on a different length scale. We will study the mechanisms and roles of the spatial spreading of Rho GTPase proteins by measuring and perturbing the spatiotemporal dynamics of Rho signaling. The specific aims of this project are to 1) establish techniques to image Rho signaling in individual spines, 2) elucidate the mechanisms and roles of spatiotemporal dynamics of Rho during synaptic plasticity, and 3) identify signaling pathways connecting calcium with Rho GTPase activation and synaptic plasticity. This study will illuminate the molecular mechanisms of morphological and functional plasticity of dendritic spines, and will provide insights into mental diseases caused by mutations in Rho signaling pathways. PUBLIC HEALTH RELEVANCE: The shape and function of synapses are regulated by signaling mediated by Rho proteins. Many forms of mental retardation and autism are caused by abnormal Rho signaling. This project will develop a novel technique to measure the activity of Rho proteins in single synapses to elucidate the mechanisms linking the activity of Rho proteins and the morphology and function of synapses. This will facilitate understanding of mental retardation and autism.